Rapid multiplex PCR and real-time TaqMan PCR assays for detection of Salmonella enterica and the highly virulent serovars Choleraesuis and Paratyphi C

Abstract

Salmonella enterica is a human pathogen with over 2,500 serovars characterized. S. enterica serovars Choleraesuis and Paratyphi C are two globally distributed serovars. We have developed a rapid molecular-typing method to detect serovars Choleraesuis and Paratyphi C in food samples by using a comparative-genomics approach to identify regions unique to each serovar from the sequenced genomes. A Salmonella-specific primer pair based on oriC was designed as an internal control to establish accuracy, sensitivity, and reproducibility. Serovar-specific primer sets based on regions of difference between serovars Choleraesuis and Paratyphi C were designed for real-time PCR assays. Three primer sets were used to screen a collection of over 100 Salmonella strains, and both serovars Choleraesuis and Paratyphi C gave unique amplification patterns. To develop the technique for practical use, its sensitivity for detection of Salmonella spp. in a food matrix was determined by spiking experiments. The technique was also adapted for a real-time PCR rapid-detection assay for both serovars Choleraesuis and Paratyphi C that complements the current procedures for Salmonella sp. isolation and serotyping.

FIG. 1.

The 461-bp PCR amplicon using the primer set ConOri-F and ConOri-R showing a representative group from the SARB and SARC of the strains in Table S1 in the supplemental material. Lane 1, 1-kb ladder.

FIG. 2.

(A) mPCR assay showing the positive identification of Salmonella spp. and serovars Choleraesuis and Paratyphi C by their specific banding patterns from DNA samples. A representative selection of other prevalent Salmonella serovars is shown. Lanes: M, 1-kb ladder; En, serovar Enteritidis; Pc, serovar Paratyphi C (SARB48 and SARB49); Tm, serovar Typhimurium; Cs, serovar Choleraesuis (SARB4, SARB6, and SC-B67); Tp, serovar Typhi (SARB63); N, negative control (null). (B) mPCR assay directly from the selective media RV and MKTTn. Lanes: M, 1-kb ladder; Pc, Cs, and Tm, serovars Paratyphi C, Choleraesuis, and Typhimurium (representative of all of the other serovars); Ec, E. coli; Pf, P. fluorescens; Lm, L. monocytogenes; N, no-template PCR control; Ns, no-initial-spike control.

FIG. 3.

(A) Sensitivity of the mPCR serovar Choleraesuis identification assay. Lane 1 had an initial spike of 250 CFU, lane 2 had 25 CFU, and lane 3 had 3 CFU. Serovar Choleraesuis-specific bands at 461 bp and 709 bp appeared in lanes 1 and 2. (B) Sensitivity of the mPCR serovar Paratyphi C identification assay. Lane 1 had an initial spike of 200 CFU, lane 2 had 20 CFU, and lane 3 had 2 CFU. Serovar Paratyphi C-specific bands appeared in lane 1, lane 2, and lane 3. There were three faint serovar Paratyphi C bands in lane 4 and a single faint band of 461 bp in lane 5. (C) Salmonella serovar Paratyphi C and serovar Choleraesuis identification assays were tested on the alternative food matrix, pasteurized milk. Lane M, marker; lane Pc, serovar Paratyphi C; lane Cs, serovar Choleraesuis; lane Tm, serovar Typhimurium; lane N, no-template PCR control.